Biomolecules, including protein, peptide, nucleic acid, oligosaccharide, glycoprotein, lipid, carbohydrate, hormones, toxins, and cells are studied by multiple analytical methods to determine their structure and function, to characterize any modifications and the effects of these modifications (e.g. structure-function relationships), and to quantify changes in the biomolecule levels and their interactions in response to growth, development, disease, treatment, and other environmental factors. These biomolecules may be present in the environment, in blood or serum, in tissue, in cells, in subcellular compartments, and/or in cellular complexes.
Many reagents and research tools have been developed to enrich biomolecules of interest for these studies. These reagents include biotin, desthiobiotin, nitrophenyl reagents, and other bioconjugation derivatives. In one example, a biomolecule is conjugated with a biotin reagent (“bait”), added to a complex sample, and then captured through the substantially irreversible 10−14 mol/L binding interaction with streptavidin-coated beads or surfaces to co-enrich other molecules (“prey”) that may bind or interact with the bait molecule. In another example, an antibody is conjugated with a biotinylating reagent so that it may be captured with an immobilized biotin-binding protein. The antibody is then added to a biological sample, wherein a target antigen is then captured from the complex protein sample with streptavidin-coated beads or surfaces for characterization or quantification.
Prior to elution of bound material, particles may be washed with non-denaturing detergents, high or low salt, pH, or solvents to reduce nonspecific background, and then the antigen can be eluted with strong acidic or basic pH conditions or denaturing concentrations of detergent. In one example, a biotinylated protein or peptide can be enriched with streptavidin coated beads, but can only be recovered through heating at 90° C. in combination with acidic buffers, organic solvents, strong detergents, and excess competitive biotin. Desthiobiotin, iminobiotin, monomeric avidin, and nitrosylated streptavidin are all reagents with lower affinity interactions, but these still require heat, extreme pH, and/or other harsh elution conditions to recover biotinylated proteins or peptides efficiently. This strategy will also capture endogenously biotinylated molecules, which may interfere with the analysis.
Alternatively, fusion proteins are expressed with N- or C-terminal affinity tags, such as 6×His, FLAG, glutathione S-transferase (GST), or hemagglutinin (HA). Each of these tags can be captured with affinity resins to purify expressed fusion proteins. In one example, a protein with a 6×His tag can be captured through the strong interaction between the imidazole ring on histidine residues in the affinity tag and a nickel or cobalt chelated immobilized metal affinity column (IMAC). This interaction is not affected by denaturing conditions, allowing aggregates of His-tagged protein to be solubilized in 8M urea denaturing conditions, captured with Ni- or Co-IMAC, refolded on column by reducing the urea concentration, and then competitively eluted with imidazole. In another example, a fusion protein expressed with GST, FLAG, or HA affinity tag can be purified with glutathione resin or immobilized anti-FLAG or anti-HA antibody resins, respectively, and then competitively eluted with free glutathione, FLAG peptide, or HA peptide. When the antigen of interest is present at a very low concentration in a complex sample, competitive and selective elution conditions may be necessary to reduce the background of non-specific biomolecules that may co-elute under harsh or denaturing conditions. Competitive elution conditions improve the quantitative recovery of the analyte and the specificity of elution of biomolecules.
The analysis of rare protein modifications and of protein-protein interactions is complicated by the low stoichiometry and transient nature of the interactions. In addition, while many modifications and interactions may be detected by antibody-based methods, the exact location and molecular characterization of these modifications or interactions are difficult to study. In one example, a protein may be nitrosylated on cysteine residues in response to oxidative stress or other environmental stimuli. While it is possible to use chemical methods to specifically biotinylate nitrosylated sites using a “biotin-switch” methodology, and antibody-based methods to capture a protein of interest and detect the presence of nitrosylation, it is very difficult to identify the site of modification and quantitatively monitor the site-specific changes under different treatment conditions or over time.
Similarly, a protein-protein interaction can be inferred by the co-immunocapture of a prey protein with a bait protein, but the molecular details of this interaction are missing without a chemical means of tagging the precise sites of interaction. In one example, a homobifunctional, amine-reactive chemical crosslinker is added to a cell lysate, a protein of interest is captured with an antibody to that protein, and other proteins that were co-enriched are assessed by resolving the enriched sample electrophoretically on a denaturing polyacrylamide gel, transferring the lane of separated proteins to a membrane, and then probing this membrane with an antibody against candidate proteins by Western blot to detect interaction and co-enrichment. In another example, the crosslinker may contain an affinity handle so that the sample may be digested and the cross-linked fragments may be enriched with the affinity handle to allow identification of the cross-linked fragments and their sites of linkage. Reagents that allow enrichment and quantitative analysis of these modifications and interactions may provide critical understanding of protein structure and interactions.
The efficiency of capture depends upon the affinity of the binding interaction, the presence of interfering molecules, and the accessibility of the binding components. Accessibility may be affected by protein folding, aggregation, complexes, and incomplete solubilization. In one example, an antibody is used to capture a protein antigen from a cell lysate. The efficiency of capture is determined in a complementary assay, such as ELISA or Western blot, by comparing samples of the starting lysate, the captured protein sample, and the depleted lysate. Because capture or depletion is often incomplete, reagent controls may be used to account for differences in capture efficiency between conditions. In one example, a version of the targeted protein is expressed or tagged with a unique dye, isotopic signature, or other modification and then spiked into all samples, captured along with the native target of interest, and then quantified to assess and normalize capture efficiency across all samples. In a similar example, multiple samples from multiple conditions are modified with a set of similar but distinct tags, the uniquely tagged samples are combined, the antigens are captured simultaneously in one reaction, and then the relative intensities of the unique tags are quantified after elution to compare protein amounts. This multiplexed capture strategy allows many samples to be enriched and analyzed simultaneously from one capture experiment. This strategy reduces the variability introduced with separate capture and elution experiments.
It is often desirable to elute a captured biomolecule or captured cell in its native form in order to preserve structural features, enzymatic function, or cellular viability. In one example, a biomolecule may be labeled with a modified biotin reagent, such as desthiobiotin, and/or captured with a modified biotin binding protein, such as monomeric avidin, which have a lower binding affinity than the native avidin-biotin interaction. After capture, the bound biomolecule may be competitively eluted with free biotin. Despite the lower affinity, this binding interaction is cooperative and high affinity, making it difficult to dissociate with high recovery without heat or extreme pH conditions. An elution reagent that can quickly and efficiently elute captured molecules under gentle conditions may better preserve the target analyte structure and function.
Similarly, after capture and elution of a molecule, it is often desirable to remove or neutralize the elution reagent without affecting sample quality or recovery. In one example, a desthiobiotin-labeled sample may be eluted from streptavidin with a solution of sodium dodecyl sulfate (SDS), a strong denaturing detergent. In some analyses, such as Western blotting, this detergent may be advantageous for solubilization and denaturation. However, in other analyses, such as mass spectrometric analysis, even low quantities of this detergent can prevent detection of the analyte and cause deterioration of the instrument performance. For standard liquid chromatography-mass spectrometric (LC-MS) analysis, it is often necessary to neutralize and remove the elution reagent in order to avoid interferences and the need to replace chromatography consumables and/or repair instrumentation.